Proteins have become commercially important as drugs that are also generally called “biologicals”. One of the greatest challenges is the development of cost effective and efficient processes for purification of proteins on a commercial scale. While many methods are now available for large-scale preparation of proteins, crude products, such as body fluids, contain not only the desired product but also impurities which are difficult to separate from the desired product. Moreover, biological sources of proteins usually contain complex mixtures of materials.
Biological sources such as cell culture supernatants of cells expressing a protein product in a recombinant way may contain less impurities, in particular if the cells are grown in serum-free medium. However, the health authorities request high standards of purity for proteins intended for human administration. In addition, many purification methods may contain steps requiring application of low or high pH, high salt concentrations or other extreme conditions that may jeopardize the biological activity of a given protein. Thus, for any protein it is a challenge to establish a purification process allowing for sufficient purity while retaining the biological activity of the protein.
Ion exchange chromatographic systems have been used widely for separation of proteins primarily on the basis of differences in charge. In ion exchange chromatography, charged patches on the surface of the solute are attracted by opposite charges attached to a chromatography matrix, provided the ionic strength of the surrounding buffer is low. Elution is generally achieved by increasing the ionic strength (i.e. conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution).
Anion exchangers can be classified as either weak or strong. The charge group on a weak anion exchanger is a weak base, which becomes de-protonated and, therefore, looses its charge at high pH. DEAE-cellulose is an example of a weak anion exchanger, where the amino group can be positively charged below pH˜9 and gradually loses its charge at higher pH values. Diethylaminoethyl (DEAE) or diethyl-(2-hydroxy-propyl)aminoethyl (QAE) have chloride as counter ion, for instance.
A strong anion exchanger, on the other hand, contains a strong base, which remains positively charged throughout the pH range normally used for ion exchange chromatography (pH 1-14). Q-sepharose (Q stands for quaternary ammonium) is an example for a strong anion exchanger.
Cation exchangers can also be classified as either weak or strong. A strong cation exchanger contains a strong acid (such as a sulfopropyl group) that remains charged from pH 1-14; whereas a weak cation exchanger contains a weak acid (such as a carboxymethyl group), which gradually loses its charge as the pH decreases below 4 or 5. Carboxymethyl (CM) and sulphopropyl (SP) have sodium as counter ion, for example.
Chromatographic systems having a hydrophobic stationary phase have also been widely employed in the purification of proteins. Included in this category are hydrophobic interaction chromatography (HIC) and reversed phase liquid chromatography (RPLC). The physicochemical basis for separation by HIC and RPLC is the hydrophobic effect, proteins are separated on a hydrophobic stationary phase based on differences in hydrophobicity.
In HIC, generally, sample molecules in a high salt buffer are loaded on the HIC column. The salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column. The more hydrophobic the molecule, the less salt needed to promote binding. Usually, a decreasing salt gradient is used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases and molecules elute from the column in order of increasing hydrophobicity. Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer. HIC is reviewed e.g. in Protein Purification, 2d Ed., Springer-Verlag, New York, pgs 176-179 (1988).
In HIC, different chromatographic supports are available carrying various ligands. The ligands differ with respect to their hydrophobicity. Commonly used hydrophobic ligands are phenyl-, butyl- or octyl- residues.
Hydrophobic charge-induction chromatography is a subset of HIC using resins carrying ligands such as 4-mercaptotheylpyridine derivatives. An example for a Hydrophobic charge-induction chromatography resin is MEP-HyperCel® (Boschetti et al., Genetic Engineering Vol. 20, No. 13, July, 2000, Boschetti and Jungbauer, 2000).
Reverse phase chromatography is a protein purification method closely related to HIC, as both are based upon interactions between solvent-accessible non-polar groups on the surface of biomolecules and hydrophobic ligands of the matrix. However, ligands used in reverse phase chromatography are more highly substituted with hydrophobic ligands than HIC ligands. While the degree of substitution of HIC adsorbents may be in the range of 10-50 μmoles/mL of matrix of C2-C8 aryl ligands, several hundred μmoles/mL of matrix of C4-C8 alkyl ligands are usually used for reverse phase chromatography adsorbents.
Hydrophobic interaction chromatography and reverse phase chromatography are also distinct in that hydrophobic interaction chromatography is performed in aqueous solvent conditions and changes in ionic strength are used to elute the column. The protein typically binds in the native state via hydrophobic groups located on the surface of the protein, and the native state is retained during the elution conditions. In contrast to this, reverse phase chromatography utilizes a hydrophobic solvent (typically acetonitrile) and the binding of a ligand is a function of the phase partition between the hydrophobic nature of the solvent and column functional group. Proteins are typically denatured to some extent in such solvents and bind due to the hydrophobic nature of the entire polypeptide sequence. Since the majority of hydrophobic groups are located in the core of globular proteins, the binding is related to the extent of denaturation of the protein and the accessibility of these groups to the column functional groups.
The Source 30RPC column is a polymeric reverse phase matrix. It is based on rigid, monosized 30 micron diameter polystyrene/divinyl benzene beads. Its characteristics can be summarized as follows: Exceptionally wide pH range (1-12), high selectivity, high chemical resistance, high capacity and high resolution at high flow rates.
A further type of chromatography widely used for protein purification is called immobilized metal ion affinity chromatography (IMAC). In 1975, Porath introduced immobilized metal ion affinity chromatography (IMAC) for fractionating proteins [J. Porath, J. Carlsson, I. Olsson, and G. Belfrage, Nature (London) 258, 598-599 (1975)]. In Porath's work, IMAC consists of derivatizing a resin with iminodiacetic acid (IDA) and chelating metal ions to the IDA-derivatized resin. The proteins are separated on the basis of their affinity for metal ions, which have been immobilized by chelation. Proteins bind to the metal ions through unoccupied coordination sites and are immobilized on the column. Since then, other ligands than IDA were used to chelate metal ions to resins. Studies with serum proteins have shown IMAC to be an extremely specific and selective separation technique [J. Porath and B. Olin, Biochemistry 22, 1621-1630 (1983)]. The adsorbent is prepared by coupling a metal chelate forming ligand, such as, iminodiacetic acid, to Sepharose or superose and treating it with a solution of one or more divalent metal ions such as Zn2+, Cu2+, Cd2+, Hg2+, Co2+, Ni2+, or Fe2+. The binding reaction is pH dependent and elution is carried out by reducing the pH and increasing the ionic strength of the buffer or by including EDTA in the buffer.
The actual mechanisms which give rise to the binding of proteins to free metal ions are not well understood and are dependent upon a number of factors, not the least of which is the conformation of the particular protein. However, when the metal ions are immobilized, at least three limiting factors come into play, namely reduced number of available coordination sites on the metal, restricted accessibility of the tethered metal to the binding sites on the protein, and, depending upon the characteristics of the resin, limited protein access to the immobilized metal ion. Thus, it is extremely difficult a priori to state which proteins will and which will not exhibit an affinity for immobilized metal ions.
Interleukin-18 binding protein (IL-18BP) is a naturally occurring soluble protein that was initially affinity purified, on an IL-18 column, from urine (Novick et al. 1999). IL-18BP abolishes IL-18 induction of IFN-γ and IL-18 activation of NF-κB in vitro. In addition, IL-18BP inhibits induction of IFN-γ in mice injected with LPS.
The IL-18BP gene was localized to the human chromosome 11, and no exon coding for a transmembrane domain could be found in the 8.3 kb genomic sequence comprising the IL-18BP gene. Four isoforms of IL-18BP generated by alternative mRNA splicing have been identified in humans so far. They were designated IL-18BP a, b, c, and d, all sharing the same N-terminus and differing in the C-terminus (Novick et al 1999). These isoforms vary in their ability to bind IL-18 (Kim et al. 2000). Of the four human IL-18BP (hIL-18BP) isoforms, isoforms a and c are known to have a neutralizing capacity for IL-18. The most abundant IL-18BP isoform, isoform a, exhibits a high affinity for IL-18 with a rapid on-rate and a slow off-rate, and a dissociation constant (Kd) of approximately 0.4 nM (Kim et al. 2000). IL-18BP is constitutively expressed in the spleen, and belongs to the immunoglobulin superfamily. The residues involved in the interaction of IL-18 with IL-18BP have been described through the use of computer modelling (Kim et al. 2000) and based on the interaction between the similar protein IL-1βwith the IL-1R type I (Vigers et al. 1997).
IL-18BP is constitutively present in many cells (Puren et al. 1999) and circulates in healthy humans (Urushihara et al. 2000), representing a unique phenomenon in cytokine biology. Due to the high affinity of IL-18BP to IL-18 (Kd=0.4 nM) as well as the high concentration of IL-18BP found in the circulation (20 fold molar excess over IL-18), it has been speculated that most, if not all of the IL-18 molecules in the circulation are bound to IL-18BP. Thus, the circulating IL-18BP that competes with cell surface receptors for IL-18 may act as a natural anti-inflammatory and an immunosuppressive molecule.
IL-18BP has been suggested as a therapeutic protein in a number of diseases and disorders, such as psoriasis, Crohn's Disease, rheumatoid arthritis, psoriatic arthritis, liver injury, sepsis, atherosclerosis, ischemic heart diseases, allergies, etc., see e.g. WO9909063, WO0107480, WO0162285, WO0185201, WO02060479, WO02096456, WO03080104, WO02092008, WO02101049, WO03013577. Given that IL-18BP is suggested as a therapeutic protein for administration e.g. to humans, there is a need for adequate amounts of IL-18BP in sufficiently high purity.
However, so far, no purification process is available that provides purified IL-18BP.